Genetic disorders have provided important model systems to identify factors and mechanisms involved in intracellular lipid metabolism and trafficking. For example, human fibroblast (Hf) cells from patients homozygous in familial hypercholesteremia have been used to elucidate the low density lipoprotein receptor pathway involved in regulation of intracellular cholesterol metabolism (Brown, M. S. and Goldstein, J. L. Science 1986 232:34–47). Niemann-Pick type C disease has also provided important insights into cholesterol metabolism. Niemann-Pick type C disease is an autosomal recessive, neurovisceral disorder that affects children who carry homozygous forms of the mutant NPC1 gene (Carstea et al. Science 1997 277:228–231) and causes death before adulthood. Hf cells from patients with Niemann-Pick type C disease have been found to accumulate LDL-derived cholesterol as unesterified cholesterol in an intracellular compartment (Pentchev et al. Proc. Natl Acad. Sci. USA 1985 82:8247–8251; Pentchev et al. FASEB J. 1987 1:40–45; and Liscum et al. J. Cell Biol. 1989 108:1625–1636).
The human NCP1 gene has been cloned, thus providing a better understanding of Niemann-Pick type C disease at the molecular level (Cartsea et al. Science (1997) Proc. Natl Acad. Sci. USA 1997 277:228–231). Final cloning work involved the identification of a 300 kb human genomic DNA containing the candidate NCP1 gene (Gu et al. Proc. Natl Acad. Sci. USA 1997 94:7378–7383). This unique DNA was identified by its ability to complement the defect of a previously isolated Chinese hamster ovary (CHO) cholesterol trafficking mutant, CT60 (Cadigan et al. J. Cell Biol. 110:295–308). The human NCP1 gene encodes an integral membrane protein with 1278 amino acids and contains the “sterol-sending domains” (Watari et al. J. Biol. Chem. 1999 274:2111861–21866) identified in several other integral membrane proteins that respond to endoplasmic reticulum (ER) cholesterol.
In mammalian cells, low density lipoprotein (LDL) binds to its receptor and internalizes and enters the endosomes/lysosomes for hydrolysis of the lipid cargo cholesteryl esters (Brown, M. S. and Goldstein, J. L. Science 1986 232:34–47). Previously Niemann Pick type C (NPC) cells were believed to be defective in the movement of LDL-derived cholesterol from the hydrolytic organelle to the plasma membrane, thereby leading to cholesterol accumulation in the lysosomes (Liscum et al. J. Cell Biol. 1989 108:1625–1636; Neufeld et al. 1996 J. Cell Biol. 271:21604–21613). Evidence at the microscopic level, however, illustrated cholesterol to accumulate in the late endosomes of NPC cells (Neufeld et al. J. Biol. Chem. 1999 274:9627–9635; Kobayashi et al. Nat. Cell Biol. 1999 1:113–118) In additional studies, the movement of LDL-derived cholesterol from the lysosomes to the plasma membrane in NPC-like cells was shown not to be defective (Lange et al. J. Biol. Chem. 1998 J. Biol. Chem 273:18915–18922). Using two independently isolated cholesterol-trafficking mutants defective in NPC1, namely CT60 and CT43, a NPC1 stable transfectant and their parental cells, 25RA CHO cells, Cruz et al. recently disclosed evidence that NPC1 is involved in post-plasma membrane cholesterol trafficking (Cruz et al. J. Biol. Chem. 2000 275(6):4013–4021). Specifically NPC1 was found to cycle cholesterol from an intracellular compartment to the plasma membrane or to the endoplasmic reticulum, but not prior to, newly hydrolyzed LDL-derived cholesterol appears in the plasma membrane (Cruz et al. J. Biol. Chem. 2000 275(6):4013–4021).